Use of cytokines and mitogens to inhibit pathological immune responses

ABSTRACT

A method for treating autoimmune disease by removing peripheral blood mononuclear cells from a patient, treating the mononuclear cells with an inhibitory composition that suppresses immunoglobulin production, and reintroducing the treated cells to the patient. The inhibitory composition can be IL-2, TGF-β, or a CD2 activator.

This application claims benefit of Provisional Application 60/064,507filed Nov. 5, 1997.

FIELD OF THE INVENTION

The field of the invention is generally related to methods of treatingantibody-mediated autoimmune diseases.

BACKGROUND OF THE INVENTION

Autoimmune diseases are caused by the failure of the immune system todistinguish self from non-self. In these diseases, the immune systemreacts against self tissues and this response ultimately causesinflammation and tissue injury. Autoimmune diseases can be classifiedinto two basic categories: antibody-mediated diseases such as systemiclupus erythematosus (SLE), pemphigus vulgaris, myasthenia gravis,hemolytic anemia, thrombocytopenia purpura, Grave's disease, Sjogren 'sdisease and dermatomyositis; and cell-mediated diseases such asHashimoto's disease, polymyositis, disease inflammatory bowel disease,multiple sclerosis, diabetes mellitus, rheumatoid arthritis, andscleroderma.

In many autoimmune diseases, tissue injury is caused by the productionof antibodies to native tissue. These antibodies are calledautoantibodies, in that they are produced by a mammal and have bindingsites to the mammals own tissue. Some of these disorders havecharacteristic waxing and waning of the amount of autobodies circulatingcausing varying symptoms over time.

Of the different types of antibody-mediated autoimmune disorders, SLE isa disorder that has been well studied and documented. SLE is a disorderof generalized autoimmunity characterized by B cell hyperactivity withnumerous autoantibodies against nuclear, cytoplasmic and cell surfaceantigens. This autoimmune disease has a multifactorial pathogenesis withgenetic and environmental precipitating factors (reviewed in Hahn, B.H., Dubois' Lupus Erythematosus, 5th Ed. (1997), pp. 69-76 (D. J.Wallace et al. eds., Williams and Wilkins, Baltimore)). Among thenumerous lymphocyte defects described in SLE is a failure of regulatoryT cells to inhibit B cell function (Horwitz, D. A., Dubois' LupusErythematosus, 5th Ed. (1997), pp. 155-194 (D. J. Wallace et al. eds.,Williams and Wilkins, Baltimore)). Regulatory T cells can down-regulateantibody synthesis by lytic or cytokine-mediated mechanisms. The latterinvolve transforming growth factor-beta (TGFβ) and other inhibitorycytokines (Wahl, S. M. (1994), J Exp Med 180:1587-190). Circulating Blymphocytes spontaneously secreting Ig are increased in patients withactive SLE (Klinman, D. M. et al. (1991), Arthritis Rheum 34:1404-1410).Sustained production of polyclonal IgG and autoantibodies in vitrorequires T cell help (Shivakumar, S. et al. (1989), J Immunol143:103-112).

Clinical manifestations of SLE include a rash (especially on the face ina “butterfly” distribution), glomerulonephritis, pleurisy, pericarditisand central nervous system involvement. Most patients are women, and arerelatively young (average age at diagnosis is 29).

The treatment of SLE depends on the clinical manifestations. Somepatients with mild clinical symptoms respond to simple measures such asnonsteroidal anti-inflammatory agents. However, more severe symptomsusually require steroids with potent anti-inflammatory andimmunosuppressive action such as prednisone. Other strongimmunosuppressive drugs which can be used are azathioprine andcyclophosphamide. The steroids and other immunosuppressive drugs haveside effects due to the global reduction of the mammal's immune system.There is presently no ideal treatment for SLE and the disease cannot becured.

Currently, considerable attention has been focused on the identity ofgenes which enhance the susceptibility or resistance to SLE, theidentification of antigenic determinants that trigger the disease, themolecular mechanisms of T cell activation which results in survival orapoptosis, cytokines which determine T cell function, and the propertiesof the autoantibody-forming B cells. Many examples of T celldysregulation in SLE have been described (reviewed in Horwitz, D. A. etal., Dubois' Lupus Erythematosus, 5th Ed. (1997), pp. 83-96 (D. J.Wallace et al. eds., Williams and Wilkins, Baltimore). Although it iswell recognized that the primary role of certain lymphocytes is todown-regulate immune responses, progress in elucidating the identity andmechanisms required for generation of these cells has been slow.

Interleukin-2 (IL-2) has previously been considered to have an importantrole in the generation of antigen non-specific T suppressor cells.Anti-IL-2 antibodies given to mice coincident with the induction ofgraft-versus-host-disease resulted in several features of SLE (Via, C.S. et al. (1993), International Immunol. 5:565-572). Whether IL-2directly or indirectly is important in the generation of suppression hasbeen controversial (Fast, L. D. (1992), J. Immunol. 149:1510-1515;Hirohata, S. et al. (1989), J. Immunol. 142:3104-3112; Baylor, C. E.(1992), Advances Exp. Med. Biol. 319:125-135). Recently, IL-2 has beenshown to induce CD8+ cells to suppress HIV replication in CD4+ T cellsby a non-lytic mechanism. This effect is cytokine mediated, but thespecific cytokine has not been identified (Kinter, A. L. et al. Proc.Natl. Acad. Sci. USA 92:10985-10989; Barker, T. D. et al. (1996), J.Immunol. 156:4478-4483). T cell production of IL-2 is decreased in SLE(Horwitz, D. A. et al. (1997), Dubois' Lupus Erythematosus, 5th Ed.(1997), pp. 83-96, D. J. Wallace et al. eds., Williams and Wilkins,Baltimore).

CD8+ T cells from subjects with SLE sustain rather than suppresspolyclonal IgG production (Linker-Israeli, M. et al. (1990), ArthritisRheum. 33:1216-1225). CD8+ T cells from healthy donors can be stimulatedto enhance Ig production (Takahashi, T. et al. (1991), Clin. Immunol.Immunopath. 58:352-365). However, neither IL-2 nor CD4+ T cells, bythemselves, were found to induce CD8+ T cells to develop strongsuppressive activity. When NK cells were included in the cultures,strong suppressive activity appeared (Gray, J. D. et al. (1994) J. Exp.Med. 180:1937-1942). It is believed that the contribution of NK cells inthe culture was to produce transforming growth factor beta (TGFβ) in itsactive form. It was then discovered that non-immunosuppressive (2-10pg/ml) concentrations of this cytokine served as a co-factor for thegeneration of strong suppressive effects on IgG and IgM production(Gray, J. D. et al. (1994) J. Exp. Med. 180:1937-1942). In addition, itis believed that NK cells are the principal source of TGFβ inunstimulated lymphocytes (Gray, J. D. et al. (1998), J. Immunol.160:2248-2254).

TGFβ is a multifunctional family of cytokines important in tissuerepair, inflammation and immunoregulation (Massague, J. (1980), Ann.Rev. Cell Biol. 6:597). TGFβ is unlike most other cytokines in that theprotein released is biologically inactive and unable to bind to specificreceptors (Sporn, M. B. et al. (1987) J. Cell Biol. 105:1039-1045). Thelatent complex is cleaved extracelluarly to release active cytokine asdiscussed below. The response to TGFβ requires the interaction of twosurface receptors (TGFβ-R1) and TGFβ-R2) which are ubiquitously found onmononuclear cells (Massague, J. (1992), Cell 69:1067-1070). Thus, theconversion of latent to active TGFβ is the critical step whichdetermines the biological effects of this cytokine.

It was found that SLE patients have decreased production of TGFβ by NKcells. Defects in constitutive TGFβ produced by NK cells as well asinduced TGFβ were documented in a study of 38 SLE patients (Ohtsuka, K.et al. (1998), J. Immunol. 160:2539-2545). Interestingly, the additionof neither recombinant IL-2 nor TNF-alpha, nor antagonism of IL-10 couldnot normalize the TGFβ defect in SLE. Decreased production of TGFβ inSLE did not correlate with activity of disease and, therefore, may be aprimary defect.

Systemic administration of Treating a SLE patient with systemic TGFβ,IL-2, or a combination of both can lead to serious side effects. Thesecytokines have numerous effects on different body tissues and are notvery safe to deliver to a patient systemically. It is, therefore, anobject of the invention to provide methods and kits for treatingmammalian cells that are responsible for controlling the regulation ofautoantibodies to increase the population of cells that down regulateauto-antibody production.

SUMMARY OF THE INVENTION

In accordance with the objects outlined herein, the present inventionprovides methods for inhibiting Ig production in a sample of ex vivoperipheral blood mononuclear cells (PBMCs) comprising adding aninhibitory composition to the cell population.

In an additional aspect, the present invention provides methods fortreating an autoimmune disorder in a patient. The methods compriseremoving peripheral blood mononuclear cells (PBMC) from the patient andtreating the cells with an inhibitory composition for a time sufficientto suppress Ig production or stimulate or induce cells to down regulateIg production. The cells are then reintroduced to the patient, with aresulting amelioration of the autoimmune symptoms. The inhibitorycomposition preferably comprises a combination of IL-2 and TGF-β.

In an additional aspect, the invention provides kits for the treatmentof an autoimmune disorder in a patient. The kits comprise a celltreatment container adapted to receive cells from a patient with anantibody-mediated autoimmune disorder and at least one dose of aninhibitory composition.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows that incubation of SLE patients PBMC with IL-2 and TGF-βdecreases spontaneous immunoglobulin production. PBMC (2×10⁵/well) werecultured in AIM-V serum free medium with or without IL-2 (10U/ml) andTGF-β (10pg/ml). After 3 days, the wells were washed three times andfresh AIM-V medium added. Supernatants were collected from the wellsafter a further 7 days and IgG content determined by an ELISA.

FIG. 2 shows that both IL-2 and TGF-β significantly decrease spontaneousIgG production. The values represent the mean ± SEM of IgG (μg/ml)produced by the 12 SLE patients PBMC cultured as described in legend toFIG. 1 except some cells were also incubated with IL-2 (10U/ml) or TGF-β(10pg/ml) only.

FIGS. 3A and 3B show that anti-TGF-β can reverse the effects of IL-2.SLE patients PBMC was cultured for three days in the presence (solidbars) or absence (spotted bars) of IL-2 (10U/ml). Included in thesecultures was medium, anti-TGF-β (10μg/ml) or control mouse IgG1(10μg/ml). After 3 days the wells were washed and fresh AIM-V mediumadded. Supernatants were collected after a further seven days andassayed for IgG (FIG. 3A) or anti-NP (FIG. 3B) content by an ELISA.

FIGS. 4A, 4B and 4C depict regulatory effects of DC8+ T cells onantibody production. (A) Synergism between NK cells and CD8+ cells inthe suppression of IgG production in a healthy subject. CD4+ cells and Bcells were stimulated with anti-CD2 and the effects of CD8+ cells and NKcells were examined. The combination of NK and CD8+ cells markedlyinhibited anti-CD2 induced IgG production we previously reported (Gray,J. D. et al. (1998), J Immunol 160:2248-2254; Gray, J. D. et al. (1994),J Exp Med 180:1937-1942). (B) NK cells and CD8+ cells enhance IgGsynthesis in SLE. CD4+ cells from a patient with active SLE and restingB cells from a healthy subject were stimulated with anti-CD2.Enhancement of IgG production by SLE CD8+ cells was markedly increasedby the addition of NK cells. (C) Cytokine normalization of CD8+ T cellfunction in SLE. In parallel with the study shown in FIG. 4B, CD4+ Tcells from this patient were stimulated with anti-CD2 in the presence orabsence of CD8+ T cells. IL-2 (1OU/ml) and/or TGF-β (2pg/ml) was addedwhere indicated. These cytokines abolished the helper effects of theseCD8+ cells and enabled them to inhibit IgG production by 75%.

FIG. 5 depicts the lymphocyte production of TGF-β1 by unstimulated andanti-CD2 stimulated cells. PBL from healthy donors and patients of SLEand RA were added to microtiter plates at 1×10⁵/well. Some wellsreceived the anti-CD2 mAbs GT2 (1:40) and T11 (1:80). After 2 days at37° C., supernatants were harvested and assayed for active and totalTGF-β1. Significant p values are indicated.

DETAILED DESCRIPTION

The present invention is directed to methods of treatingantibody-mediated autoimmune disorders, such as systemic lupuserythematosus (SLE), by removing cells from a patient and treating themwith a composition that will down-regulate B cell hyperactivity and thusinhibit the production of Ig antibodies, including autoantibodies, toameliorate the symptoms of the autoimmune disorder. This strategy isunlike almost all other treatment modalities currently in use which areeither anti-inflammatory or immunosuppressive. Commonly usedcorticosteroids suppress cytokine production and block the terminalevents which cause tissue injury, but generally do not alter theunderlying autoimmune response. Cytotoxic drugs or experimentalgenetically engineered biologicals such as monoclonal antibodies mayalso deplete specific lymphocyte populations or interfere with theirfunction. These drugs are generally only moderately successful and havesevere adverse side effects. Certain cytokines have been givensystemically to patients, but these agents also have broad actions withassociated serious adverse side effects.

By contrast, the strategy of the present invention is to produceremission by restoring normal regulatory cell function and, thus,“resetting” the immune system. Another significant potential advantageof this strategy is a low probability of serious adverse side effects.Since only trace amounts of inhibitory compositions such as cytokineswill be returned to the patient, there should be minimal toxicity.

Circulating B lymphocytes spontaneously secreting IgG are increased inpatients with active SLE (Blaese, R. M., et al. (1980), Am J. Med69:345-350; Klinman, D. M. et al. (1991) Arthritis Rheum 34: 1404-1410).Sustained production of polyclonal IgG and autoantibodies in vitrorequires T cell help (Shivakumar, S. et al. (1989), J Immunol143:103-112). Previous studies of T cell regulation of spontaneous IgGproduction shows that while CD8+ T cells inhibit antibody production inhealthy individuals, in SLE these cells support B cell function instead(Linker-Israeli, M. et al. (1990), Arthritis Rheum 33:1216-1225).

Accordingly, the present invention is drawn to methods of treatingantibody-mediated autoimmune diseases that comprise removing peripheralblood mononuclear cells (PBMCs) from the patient with the autoimmunedisease and treating the cells with an inhibitory composition.

Without being bound by theory, it appears that there are several waysthe methods of the invention may work. First of all, the treatment ofthe cells by an inhibitory composition leads to the direct suppressionof Ig production in the treated cells, which can lead to amelioration ofautoimmune symptoms. Alternatively or additionally, the treatment of thecells induces regulatory cells to down regulate Ig production in othercells. Ig in this context includes all forms of Ig, including IgM, IgG,IgE, etc. The net result is a decrease in the amount of Ig in thesystem.

Thus, the present invention restores the capacity of peripheral blood Tcells from patients with autoimmune disorders to down regulate antibodyproduction by treating them with an inhibitory composition ex vivo.

Accordingly, the present invention provides methods of treatingantibody-mediated autoimmune disorders in a patient. By“antibody-mediated autoimmune diseases” herein is meant a disease inwhich individuals develop antibodies to constituents of their own cellsor tissues. Antibody-mediated autoimmune diseases include, but are notlimited to, systemic lupus erythematosus (SLE), pemphigus vulgaris,myasthenia gravis, hemolytic anemia, thrombocytopenia purpura, Grave'sdisease, dermatomyositis and Sjogren's disease. The preferred autoimmunedisease for treatment using the methods of the invention is SLE.

By “treating” an autoimmune disorder herein is meant that at least onesymptom of the autoimmune disorder is ameliorated by the methodsoutlined herein. This may be evaluated in a number of ways, includingboth objective and subjective factors on the part of the patient. Forexample, immunological manifestations of disease can be evaluated; forexample, the level of spontaneous Ig antibody and autoantibodyproduction, particularly IgG production in the case of SLE, is reduced.Total Ig antibody levels may be measured, or autoantibodies, including,but not limited to, anti-double-stranded DNA (ds DNA) antibodies,anti-nucleoprotein antibodies, anti-Sm, anti-Rho, and anti-La. Physicalsymptoms may be altered, such as the disappearance or reduction in arash in SLE. Renal function tests may be performed to determinealterations; laboratory evidence of tissue damage relating toinflammation may be evaluated. Decreased levels of circulating immunecomplexes and levels of serum complement are further evidence ofimprovement. In the case of SLE, a lessening of anemia may be seen. Theability to decrease a patient's otherwise required drugs such asimmunosuppressives can also be an indication of successful treatment.Other evaluations of successful treatment will be apparent to those ofskill in the art of the particular autoimmune disease.

By “patient” herein is meant a mammalian subject to be treated, withhuman patients being preferred. In some cases, the methods of theinvention find use in experimental animals, in veterinary application,and in the development of animal models for disease, including, but notlimited to, rodents including mice, rats, and hamsters; and primates.

The methods provide for the removal of blood cells from a patient. Ingeneral, peripheral blood mononuclear cells (PBMCs) are taken from apatient using standard techniques. By “peripheral blood mononuclearcells” or “PBMCs” herein is meant lymphocytes (including T-cells,B-cells, NK cells, etc.) and monocytes. As outlined more fully below, itappears that the main effect of the inhibitory composition is to enableCD8+ T cells to suppress IgG production. Accordingly, the PBMCpopulation should comprise CD8+ T cells. Preferably, only PBMCs aretaken, either leaving or returning substantially all of the red bloodcells and polymorphonuclear leukocytes to the patient. This is done asis known in the art, for example using leukophoresis techniques. Ingeneral, a 5 to 7 liter leukophoresis step is done, which essentiallyremoves PBMCs from a patient, returning the remaining blood components.Collection of the cell sample is preferably done in the presence of ananticoagulant such as heparin, as is known in the art.

In general, the sample comprising the PBMCs can be pretreated in a widevariety of ways. Generally, once collected, the cells can beadditionally concentrated, if this was not done simultaneously withcollection or to further purify and/or concentrate the cells. The cellsmay be washed, counted, and resuspended in buffer.

The PBMCs are generally concentrated for treatment, using standardtechniques in the art. In a preferred embodiment, the leukophoresiscollection step results a concentrated sample of PBMCs, in a sterileleukopak, that may contain reagents and/or doses of the inhibitorycomposition, as is more fully outlined below. Generally, an additionalconcentration/purification step is done, such as Ficoll-Hypaque densitygradient centrifugation as is known in the art.

In a preferred embodiment, the PBMCs are then washed to remove serumproteins and soluble blood components, such as autoantibodies,inhibitors, etc., using techniques well known in the art. Generally,this involves addition of physiological media or buffer, followed bycentrifugation. This may be repeated as necessary. They can beresuspended in physiological media, preferably AIM-V serum free medium(Life Technologies) (since serum contains significant amounts ofinhibitors) although buffers such as Hanks balanced salt solution (HBBS)or physiological buffered saline (PBS) can also be used.

Generally, the cells are then counted; in general from 1×10⁹ to 2×10⁹white blood cells are collected from a 5-7 liter leukophoresis step.These cells are brought up roughly 200 mls of buffer or media.

In a preferred embodiment, the PBMCs may be enriched for one or morecell types. For example, the PBMCs may be enriched for CD8+ T cells orCD4+ T cells. This is done as is known in the art, as described in Grayet al. (1998), J. Immunol. 160:2248, hereby incorporated by reference.Generally, this is done using commercially available immunoabsorbentcolumns, or using research procedures (the PBMCs are added to a nylonwool column and the eluted, nonadherent cells are treated withantibodies to CD4, CD16, CD11b and CD74, followed by treatment withimmunomagnetic beads, leaving a population enriched for CD8+ T cells).

Once the cells have undergone any necessary pretreatment, the cells aretreated with an inhibitory composition. By “treated” herein is meantthat the cells are incubated with the inhibitory composition for a timeperiod sufficient to develop the capacity to inhibit Ig and autoantibodyproduction, particularly when transferred back to the patient. Theincubation will generally be under physiological temperature. As notedabove, this may happen as a result of direct suppression of Igproduction by the treated cells, or by inducing regulatory cells to downregulate the production of Ig in the patient's lymphoid organs.

By “inhibitory composition” or “Ig production inhibitor composition” or“humoral inhibitor composition” herein is meant a composition that cancause inhibition of spontaneous Ig and autoantibody production.Generally, these compositions are cytokines. Suitable inhibitorycompositions include, but are not limited to, IL-2, TGF-β, and CD2activators, including anti-CD2 antibodies and the CD2 ligand, LFA-3, aswell as mixtures or combinations of these. A preferred inhibitorycomposition is a mixture of IL-2 and TGF-β.

The concentration of the inhibitory composition will vary on theidentity of the composition. In a preferred embodiment, TFG-β is used asthe inhibitory composition. By “transforming growth factor -β” or“TGF-β” herein is meant any one of the family of the TGF-βs, includingthe three isoforms TGF-β1, TGF-β2, and TGF-β3; see Massague, J. (1980),J. Ann. Rev. Cell Biol 6:597. Lymphocytes and monocytes produce the β1isoform of this cytokine (Kehrl, J. H. et al. (1991), Int J Cell Cloning9: 438-450). The TFG-β can be any form of TFG-β that is active on themammalian cells being treated. In humans, recombinant TFG-β is currentlypreferred. A preferred human TGF-β can be purchased from GenzymePharmaceuticals, Farmington, Mass., In general, the concentration ofTGF-β used ranges from about 2 picograms/ml of cell suspension to about2 nanograms, with from about 10 pg to about 500 pg being preferred, andfrom about 50 pg to about 150 pg being especially preferred, and 100 pgbeing ideal.

In a preferred embodiment, IL-2 is used as the inhibitory composition.The IL-2 can be any form of IL-2 that is active on the mammalian cellsbeing treated. In humans, recombinant IL-2 is currently preferred.Recombinant human IL-2 can be purchased from Cetus, Emeryville, Calif.In general, the concentration of IL-2 used ranges from about 1 Unit/mlof cell suspension to about 100 U/ml, with from about 5 U/ml to about 25U/ml being preferred, and with 10 U/ml being especially preferred. In apreferred embodiment, IL-2 is not used alone.

In a preferred embodiment, a CD2 activator such as anti CD2 antibodiesor the CD2 ligand LFA-3 are used as the inhibitory composition. CD2 is acell surface glycoprotein expressed by T lymphocytes. By “CD2 activator”herein is meant compound that will initiate the CD2 signaling pathway. Apreferred CD2 activator comprises anti CD2 antibodies (OKT11, AmericanType Culture Collection, Rockville Md.). In general, the concentrationof CD2 activator used will be sufficient to induce the production ofTGF-β. The concentration of anti CD2 antibodies used ranges from about 1ng/ml to about 10 μg/ml, with from about 10 ng/ml to about 100 ng/mlbeing especially preferred.

In addition to treatment with an inhibitory composition, in someembodiments it is desirable to use a mitogen to activate the cells; thatis, many resting phase cells do not contain large amounts of cytokinereceptors. The use of a mitogen such as Concanavalin A can allow thestimulation of the cells to produce cytokine receptors, which in turnmakes the methods of the invention more effective. When a mitogen suchas ConA is used, it is generally used as is known in the art, atconcentrations ranging from 1 μg/ml to about 10 μg/ml is used. Inaddition, it may be desirable to wash the cells with components toremove the ConA, such as α-methyl mannoside, as is known in the art.

The inhibitory composition is incubated with the cells for a period oftime sufficient to cause an effect. In a preferred embodiment, treatmentof the cells with the inhibitory composition is followed by immediatetransplantation back into the patient. Accordingly, in a preferredembodiment, the cells are incubated with the inhibitory composition forfrom about 12 to about 120 hours, with from about 24 to about 72 hoursbeing preferred, and 48 hours being particularly preferred.

In one embodiment, the cells are treated for a period of time, washed toremove the inhibitory composition, and may be reincubated. Beforeintroduction into the patient, the cells are preferably washed asoutlined herein to remove the inhibitory composition. Furtherincubations for testing or evaluation may also be done, ranging in timefrom a few hours to several days. If evaluation of Ig production priorto introduction to a patient is desirable, the cells will be incubatedfor several days to allow Ig production (or lack thereof) to occur.

Once the cells have been treated, they may be evaluated or tested priorto autotransplantation back into the patient. For example, a sample maybe removed to do: sterility testing; gram staining, microbiologicalstudies; LAL studies; mycoplasma studies; flow cytometry to identifycell types; functional studies, etc. Similarly, these and otherlymphocyte studies may be done both before and after treatment.

In a preferred embodiment, the quantity or quality, i.e. type, of Igproduction, may be evaluated. Thus, for example, the total levels of Igmay be evaluated, or the levels of specific types of Igs, for example,IgG, IgM, etc.; IgG anti-DNA autoantibodies, anti-nucleoprotein (NP)antibodies, etc.

In a preferred embodiment, the levels of Ig, particularly IgG, aretested using well known techniques, including ELISA assays, as describedin Abo et al. (1987), Clin. Exp. Immunol. 67:544 and Linker-Israeli etal. (1990), Arthritis Rheum 33:1216, both of which are hereby expresslyincorporated by reference. These techniques may also be used to detectthe levels of specific antibodies, such as autoantibodies.

In a preferred embodiment, the treatment results in a significantdecrease in the amount of IgG and autoantibodies produced, with adecrease of at least 10% being preferred, at least 25% being especiallypreferred, and at least 50% being particularly preferred. In manyembodiments, decreases of 75% or greater are seen.

In a preferred embodiment, prior to transplantation, the amount of totalor active TGF-β can also be tested. As noted herein, TGF-β is made as alatent precursor that is activated post-translationally.

After the treatment, the cells are transplanted or reintroduced backinto the patient. This is generally done as is known in the art, andusually comprises injecting or introducing the treated cells back intothe patient, via intravenous administration, as will be appreciated bythose in the art. For example, the cells may be placed in a 50 mlFenwall infusion bag by injection using sterile syringes or othersterile transfer mechanisms. The cells can then be immediately infusedvia IV administration over a period of time, such as 15 minutes, into afree flow IV line into the patient. In some embodiments, additionalreagents such as buffers or salts may be added as well.

After reintroducing the cells into the patient, the effect of thetreatment may be evaluated, if desired, as is generally outlined above.Thus, evaluating immunological manifestations of the disease may bedone; for example the titers of total Ig or of specific immunoglobulins,renal function tests, tissue damage evaluation, etc. may be done.

The treatment may be repeated as needed or required. For example, thetreatment may be done once a week for a period of weeks, or multipletimes a week for a period of time, for example 3-5 times over a two weekperiod. Generally, the amelioration of the autoimmune disease symptomspersists for some period of time, preferably at least months. Over time,the patient may experience a relapse of symptoms, at which point thetreatments may be repeated.

In a preferred embodiment, the invention further provides kits for thepractice of the methods of the invention, i.e., the incubation of thecells with the inhibitory compositions. The kit may have a number ofcomponents. The kit comprises a cell treatment container that is adaptedto receive cells from a patient with an antibody-mediated autoimmunedisorder. The container should be sterile. In some embodiments, the celltreatment container is used for collection of the cells, for example itis adaptable to be hooked up to a leukophoresis machine using an inletport. In other embodiments, a separate cell collection container may beused.

The form and composition of the cell treatment container may vary, aswill be appreciated by those in the art. Generally the container may bein a number of different forms, including a flexible bag, similar to anIV bag, or a rigid container similar to a cell culture vessel. It may beconfigured to allow stirring. Generally, the composition of thecontainer will be any suitable, biologically inert material, such asglass or plastic, including polypropylene, polyethylene, etc. The celltreatment container may have one or more inlet or outlet ports, for theintroduction or removal of cells, reagents, inhibitory compositions,etc. For example, the container may comprise a sampling port for theremoval of a fraction of the cells for analysis prior to reintroductioninto the patient. Similarly, the container may comprise an exit port toallow introduction of the cells into the patient; for example, thecontainer may comprise an adapter for attachment to an IV setup.

The kit further comprises at least one dose of an inhibitorycomposition. “Dose” in this context means an amount of the inhibitorycomposition such as cytokines, that is sufficient to cause an effect. Insome cases, multiple doses may be included. In one embodiment, the dosemay be added to the cell treatment container using a port;alternatively, in a preferred embodiment, the dose is already present inthe cell treatment container. In a preferred embodiment, the dose is ina lyophilized form for stability, that can be reconstituted using thecell media, or other reagents.

In some embodiments, the kit may additionally comprise at least onereagent, including buffers, salts, media, proteins, drugs, etc. Forexample, mitogens can be included. In some embodiments, the kit mayadditional comprise written instructions for using the kits.

The following examples serve to more fully describe the manner of usingthe above-described invention, as well as to set forth the best modescontemplated for carrying out various aspects of the invention. It isunderstood that these examples in no way serve to limit the true scopeof this invention, but rather are presented for illustrative purposes.All references cited herein are incorporated by reference in theirentirety.

EXAMPLES Example 1 Treatment of PBMCs with a Mixture of IL-2 and TFG-β

Example 1 shows that the relatively brief treatment of PBMCs from SLEpatients with IL-2 and TFG-β can result in the marked inhibition ofspontaneous polyclonal IgG and autoantibody production. As discussedbelow, PBMC from 12 patients with active SLE were exposed to IL-2 withor without TGF-β for 3 days, washed and cultured seven more days. Themean decrease in IgG secretion was 79%. The strongest inhibitory effectwas observed in cases with the most marked B cell hyperactivity.Spontaneous production of anti-nucleoprotein (NP) antibodies wasobserved in 4 cases and cytokine treatment of PBMC decreasedautoantibody production by 50 to 96%. IL-2 inhibited Ig production byeither TGF-β-dependent or independent mechanisms in individual patients.In a study of anti-CD2 stimulated IgG production in a patient withactive SLE, we documented that IL-2 and TGF-β can reverse the enhancingeffects of CD8+ T cells on IgG production and induce suppressiveactivity instead.

Methods

Study Subjects for Spontaneous Ig Synthesis

Twelve subjects were chosen with a diagnosis of SLE that fulfilled ARAcriteria for the classification of SLE (Arnett, F. C. et al. (1998),Arthritis Rheum 31: 315-324). These patients were all women, 8 Hispanic,2 African American, and 2 Asian. The age of each patient and duration ofdisease is shown in Table 1. Five patients were hospitalized and 7 wereoutpatients. Those patients who were receiving corticosteroids orantimalarials are also indicated. 8 patients were untreated. Diseaseactivity was assessed with SLAM (Liang, M. H. et al. (1989), ArthritisRheum 32:1107-1118) and SLEDAI (Bombardier, C. et al. (1992), ArthritisRheum 35:630-640) indices with mean values of 16.5 and 13.4respectively.

TABLE 1 Profile of SLE Patients Case SEX Age Ethnicity DurationMedications SLAM SLEDAI IgG(μ/ml) 1 F 18 AA 3 yr Nil 13 9 13.7 2 F 37 H6 mo Nil 23 13 13.0 3 F 29 H 1 yr Nil 15 6 2.6 4 F 32 AA 4 yr Pred 5 mg9 6 2.5 Ohchlor 400 mg 5 F 57 A 5 mo Nil 24 19 2.2 6 F 55 H 5 mo Nil 2322 1.5 7 F 27 H 3 yr Pred 20mg 13 17 1.0 Ohchlor 400 mg 8 F 21 H 2 yrNil 18 13 1.0 9 F 36 H 15 yr Pred 20 mg 14 8 0.8 Ohchlor 400 mg Aza 25mg 10 F 41 A 4 yr Nil 15 16 0.5 11 F 20 H 6 yr Pred 25 mg 11 16 0.4 12 F25 H 1 yr NiI 21 16 0.4

Reagents

Recombinant TGF-β and monoclonal anti-TGF-β (1D11.16) antibody, a murineIgG1, were kindly provided by Dr. Bruce Pratt (Genzyme Pharmaceuticals,Farmington, Mass.). Recombinant IL-10 and monoclonal anti-IL-10(JES3-19F1) antibody, and control rat IgG2a, were kindly provided by Dr.Satwant Narula (Schering Plough Pharmaceuticals, Kenilworth, N.J.).Control murine IgG1 myeloma protein was purchased from Calbiochem, SanDiego, Calif. Recombinant human IL-2 was purchased from Chiron,Emmeryville, Calif. Anti-CD2 secreting hybridomas antibodies used OKT11were obtained from the American Type Culture Collection (ATCC),Rockville, Md. and GT2 was generously provided by A. Bernard, Nice,France). Other antibodies included: anti-CD4 (OKT4, ATCC), anti-CD8(OKT8, ATCC; CD8, Dako, Carpenteria, Calif.), anti-CD11b (OKM1, ATCC),anti-CD16 (3G8), kindly provided by J. Unkeless, New York, N.Y.);anti-CD20 (Leu 16, Becton Dickinson, San Jose, Calif.) and anti-CD74(L243, ATCC).

Isolation of Blood Mononuclear Cells

Peripheral blood mononuclear cells (PBMC) were prepared from heparinizedvenous blood by Ficoll-Hypaque (Pharmacia, Piscataway, N.J.) densitygradient centrifugation. The mononuclear cells were washed in PBS with5mM EDTA (Life Technologies, Grand Island, N.Y.) to remove platelets,which are a rich source of TGF-β.

Cell Culture Procedures

Procedures for cell cultures have been described previously (Wahl, S. M.(1994), J Exp Med 180:1587-1590; Gray, J. D. et al. (1998), J Immunol160:2248-2254). In brief, 2×10⁵ of PBMC were cultured in serum-freeAIM-V culture medium ( Life Technologies) in the wells of 96-well flatbottom microtiter plate with or without the indicated cytokines. Afterthree days of culture, the PBMC were washed three times then freshserum-free medium was added. After a further 7 days at 37° C.,supernatants were harvested and assayed for total IgG and autoantibodiesreactive with calf thymus nucleoprotein (NP) by a solid phaseenzyme-linked immunoadsorbant assay (ELISA), as described previously(Linker-Israeli, M. et al. (1990), Arthritis Rheum 33:1216-1225). Theoptical density (OD) readings were transformed into units/ml (U/ml) froma standard curve using positive and negative standards. Supernatantsfrom PBMC culture of SLE patients (with high titers of anti-NPantibodies) and normal individuals were used as controls.

Statistical Analysis

The data were analyzed using Graph Pad, Prism software (San Diego,Calif.). We used analysis of variance (ANOVA) after log transformationof the data and the non-parametric Mann-Whitney test.

Anti-CD2 Induced IgG Synthesis

The effects of CD8+ T cells cultured with or without NK cells onanti-CD2 stimulated CD4+ T cells and B cells was examined in a patientwith SLE in a normal control. CD4+ and CD8+ cells were prepared fromnylon non-adherent lymphocytes by negative selection usingimmunomagnetic beads as described previously (Gray, J. D. et al. (1998),J lmmunol 160:2248-2254). For CD4+ cells the nylon non-adherent cellswere stained with antibodies to CD8, CD16, CD11b and CD74. The sameantibodies were used to obtain CD8+ cells except that CD4 wassubstituted for CD8. Purity of CD4+ cells was 95% and CD8+ cells 89%. Toobtain NK cells, PBMC were added to a nylon wool column and the eluted,non-adherent cells were immediately rosetted with AET treated sheep redblood cells. The non-rosetting fraction was then stained with anti-CD3and anti-CD74 (anti-HLA-DR) antibodies and depleted of reacting cellsusing immunomagnetic beads (Dynal). This resultant population contained98% CD56+ and <0.5% CD3+ and <0.5% CD20+ lymphocytes. Since SLE B cellsspontaneously secrete large amounts of IgG and because of the largeamount of blood needed to prepare sufficient numbers of B cells forthese studies, we substituted resting B cells from a healthy donor forpatient B cells in this study. To obtain B cells, nylon wool adherentcells were immediately rosetted with SRBC to remove any T cells, andtreated with 5 mM L-leucine methyl ester for complete removal ofmonocytes and functional NK cells. The resulting population was >92%CD20+ and <0.5% CD3+.

Results

In 12 patients studied, spontaneous IgG ranged from 0.4 to 13.7 μg/ml(FIG. 1). Exposure of PBMC to IL-2 ± TGF-β for 72 hours decreased IgGsynthesis in 8 of 12 cases studied by at least 50% (mean decrease 79%,p=0.008, Mann Whitney). The most dramatic decreases were observed incases with the most marked B cell hyperactivity. The correlation betweenthe amount of IgG secreted and percent inhibition by IL-2 and TGF-β wasr=0.647, p=0.02.

We compared the effects of IL-2 and TGF-β alone to the combination ofIL-2 and TGF-β. FIG. 2 shows that each of these cytokines also inhibitedIL-2 production. However, after log transformation to achieve a normaldistribution of the data and applying the Bonnferoni correction formultiple comparisons, analysis of variance revealed that only thecombination of IL-2 and TGF-β resulted in significant inhibition(p=0.05).

IL-10 production is increased in SLE (Llorente, L. et al. (1993), EurCytokine Network 4.421-427) and this cytokine can inhibit production ofboth IL-2 and TGF-β. In 9 cases we also assessed the effect ofanti-lL-10, but only a modest decrease of IgG synthesis was observed insome subjects and this difference was not statistically significant.Similarly, TNFα production is also decreased in a subset of patientswith SLE (Jacob, C. O. et al. (1990), Proc Nati Acad Sci 87:1233-1237).Although this cytokine also increases the production of active TGF-β(Ohtsuka, K. et al. (198), J Immunol 160:2539-2545), the addition ofTNFα to the cultures had minimal effects (results not shown).

We also examined SLE PBMC for spontaneous production of anti-NPautoantibodies and found significant titers in 4 cases. In all casesexposure of PBMC to either IL-2 or IL-2 and TGF-β inhibited anti-NPproduction by at least 50 percent. TGF-β by itself was ineffective(Table 2). In these cases the effects of IL-2 by itself was equivalentto that the combination of IL-2 and TGF-β.

TABLE 2 Effect of treating PBMC with IL-2 and TGF-β on SpontaneousAutoantibody production in SLE Anti-nucleoprotein antibody (U/ml)Cytokine treatment Case A: Case B: Case C Case D Nil 308 (100)* 312(100) 25 (100) 73 (100) TGF-β (10 pg/ml) 282 (92) 298 (96) 26 (104) NDIL-2 & TGF-β  29 (10)  14 (4.5) 12 (48) 35 (48) IL-2  23 (7.5)  10 (3)11 (44) ND *Percent of baseline values

PBMC from SLE patients were exposed to IL-2 (10 u/ml) and TGF-β(10pg/ml) for 72 hours. The cells were washed and cultured for sevenadditional days. Anti-NP released into the supernatants was measured byan ELISA.

Previously we have reported that IL-2 increases the production ofbiologically active TGF-β (Ohtsuka, K. et al. (1998), J Immunol160:2539-2545). It was, therefore, possible that al least some of theeffects of IL-2 on spontaneous Ig synthesis were mediated by TGF-β. Thispossibility was investigated by determining whether the effects of IL-2could be reversed by an anti-TGF-β neutralizing antibody. In the exampleshown in FIG. 3A, the addition of anti-TGF-β did not affect spontaneousIgG synthesis. Antagonism of TGF-β, however, did abolish the inhibitoryeffects of IL-2 on IgG synthesis. PBMC from this patient (Case C inTable 2) also spontaneously produced anti-NP antibody. Here alsoanti-TGF-β abolished the inhibitory effects of IL-2 on anti-NPproduction (FIG. 3B). In this subject, therefore, the inhibitory effectsof IL-2 on spontaneous IgG and autoantibody synthesis were mediated byTGF-β. This effect of anti-TGF-β was documented in 4 of 8 cases studied.Thus, the inhibitory effects of IL-2 could either be TGF-β-dependent orindependent. Examples of each effect are shown in Table 3.

TABLE 3 Effect of IL-2 and TGF-β on Spontaneous IgG Synthesis in SLEPatient B: Patient A: TGF-β independent TGF-β dependent inhibitioninhibition Cytokines Added G (μgm/ml) IgG (μgm/ml) Medium only  2.5(100)* 2.6 (100) TGF-β (10 pg/ml)  1.4 (56) 2.5 (96) IL-2 & TGF-β  0.4(16) 0.5 (19) IL-2 & anti-TGF-β 11.6 (464) 0.5 (19) IL-2 & IgG1  3.6(144) 0.6 (23) *Percent of baseline IgG synthesis

We had the opportunity to repeat the study of on SLE patient 28 daysafter initiation of steroid therapy (Table 4). Before treatmentspontaneous IgG synthesis was greater than 2 μg/ml of IgG. Exposure ofPBMC to IL-2 markedly inhibited IgG production and TGF-β had a moderateeffect. Following corticosteroid therapy, spontaneous IgG productiondecreased by 75%. As before, exposure of PBMC to IL-2 ± TGF-β decreasedIgG production by 50%. However, this inhibition was reversed byanti-TGF-β. Here again, this effect of IL-2 could be explained byupregulation of endogenous active TGF-β.

TABLE 4 Effect of Corticosteroid Therapy on Spontaneous IgG Synthesis inSLE Before Treatment After Treatment Cytokines Added Day 0 Day 28 Nil2.2 0.6 TGF-β (10 pg/ml) 1.2 0.4 IL-2 (10 U/ml) 0.4 0.3 IL-2 & TGF-β 0.70.3 IL-2 & anti-TGF-β ND 0.8 IL-2 & IgG1 ND 0.6 *Percent of baseline IgGsynthesis

In view of our previous studies in healthy subjects that IL-2 and TGF-βcan induce activated CD+ T cells to down-regulate Ig production, weattempted to isolate and treat CD8+ T cells from SLE patients in thisstudy. These experiments were unsuccessful because of the markedvariability of spontaneous Ig synthesis and the large amount of bloodrequired from patients with active SLE for cell separation procedures.However, we were able to obtain enough blood from one patient withactive SLE to investigate the effect of IL-2 and TGF-β on CD8+ T cellmodulation of anti-CD2 induced IgG synthesis. We have recently reportedthat unlike anti-CD3, a mitogenic combination of anti-CD2 monoclonalantibodies did not induce PBL to produce IgG (Gray, J. D. et al. (1998),J Immunol 160:2248-2254). This was because anti-CD2 stimulated NK cellsto produce TGF-β, which in turn induced CD8+ T cells to down-regulate Igproduction (Gray, J. D. et al. (1998), J Immunol 160:2248-2254). In thispatient, as we have reported previously (Gray, J. D. et al. (1994), JExp Med 180:1937-1942), CD8+ T cells enhanced IgG synthesis and thisenhancement was markedly potentiated by the combination of NK cells andCD8+ T cells (FIG. 4A). By contrast IL-2 and TGF-β abolished the helpereffects of SLE CD8+ T cells and enabled these cells to suppress IgGproduction. This inhibitory effect of IL-2 and TGF-β was dependent uponthe presence of CD8+ T cells. (FIG. 4B). Thus, evidence has beenobtained that the effects of IL-2 and TGF-β can be mediated by CD8+ Tcells.

These studies demonstrate that a short exposure of PBMC to IL-2 andTGF-β can greatly decrease subsequent spontaneous polyclonal IgG andautoantibody production in SLE, especially in patients with severedisease and marked B cell hyperactivity. This study confirms previousreports indicating that IL-2 can inhibit antibody production (Hirohata,S. et al. (1989), J Immunol 142: 3104-3112 and Fast, L. D. (1992), JImmunol 149:1510-1515) and reveals that picomolar concentrations ofTGF-β can contribute to this down-regulation. In the group of 12patients studied, the inhibitory effect of IL-2 and TGF-β on polyclonalIgG synthesis was greater than the effect of IL-2 alone. However, theinhibitory effects of IL-2 were heterogeneous. In 4 of 8 cases studied,the inhibition was TGF-β-dependent in that a neutralizing anti-TGF-β mAbabolished the effect. In the remaining cases the down-regulatory effectsof IL-2 were TGF-β-independent. Similarly, both TGF-β-dependent andindependent inhibition of spontaneous anti-NP autoantibody productionwas documented. We also investigated the effects of antagonizing theIL-10 and adding TNF-α because of previously described abnormalities inthe production of these cytokines in SLE (Llorente L. et al. (1993), EurCytokine Network 4:421-427; Jacob, C. O. et al. (1990), Proc Natl AcadSci 87:1233-1237).

These procedures, however, had minimal effects on spontaneous Igsynthesis where lymphocytes had been activated previously.

Others have reported that the degree of B cell hyperactivity in SLEcorrelates with disease activity (Blaese, R. M. et al. (1980), Am J Med69:345-350; Klinman, D. M. et al. (1991), Arthritis Rheum 34:1404-1410).This was not the case in the present study, possibly because ofconcurrent drug therapy. In general, those patients with markedspontaneous Ig synthesis were untreated whereas those with less B cellactivity were currently receiving prednisone. We presented one casewhere spontaneous IgG synthesis decreased markedly after corticosteroidtherapy was begun. This patient's B cells had also been secretinganti-NP antibody before treatment, and production of this autoantibodybecame undetectable after steroid therapy (result not shown).

TGF-β consists of a multifunctional family of cytokines important intissue repair, inflammation and immunoregulation (Massague, J. (1990),Annu Rev Cell Biol 6597-641). TGF-β is different from most othercytokines in that it is secreted as an inert precursor molecule andconverted to its biologically active form extracellularly (Massague, J.(1990), Annu Rev Cell Biol 6597-641; Flaumenhaft, R. et al. (1993), AdvPharmacol 24:51-76). Regulatory T cells in various experimentalautoimmune models such as experimental autoimmune encephalitis (Weiner,H. L. et al. (1994), Annu Rev Immunol 12:809-837) and colitis (Neurath,M. F. et al. (1996), J Exp Med 183:2605-2516) produce this cytokine.TGF-β is immunosuppressive in nanomolar concentrations and can inhibit Tand B cell proliferation, NK cell cytotoxic activity and the generationof T cell cytotoxicity (Letterio, J. J. et al. (1998), Ann Rev Immunol16:137-162). By contrast, TGF-β has been reported to promote the growthof murine CD4+ cells and CD8+ cells (Kehrl, J. H. et al. (1986), J ExpMed 163:1037-1050; Lee, H. M. et al. (1993), J Immunol 151:668-677) andcan promote B cell differentiation (Van Vlasselaer, P. et al. (1992), JImmunol 148:2062-2067).

In our previous studies with lymphocytes from healthy subjects togenerate regulatory T cells, the picomolar concentrations of TGF-β usedwere smaller than that required for inhibition of T or B cell function(Gray, J. D. et al. (1998), J Immunol 160:2248-2254; Gray, J. D. et al.(1994), J Exp Med 180:1937-1942). Similar concentrations were used inthe present studies with SLE patients and TGF-β by itself had modestinhibitory effects on Ig synthesis. As before, a combination of IL-2 ofTGF-β produced the most potent inhibition. In our previous studies, thiseffect was mediated by CD8+ T cells.

IL-2 has well established effects on the induction of T suppressor cellactivity (Hirohata, S. et al. (1989), J Immunol 142:3104-3112; Fast, L.D. J Immunol 149:1510-1515), but whether these effects are direct orindirect is unclear. In mice deletion of the IL-2 gene results inmassive lymphoproliferation and autoimmune disease (Sadlack, B. et al.(1995), Eur J Immunol 25:3053-3059). In SLE, a negative correlation wasreported between IL-2 levels and B cell hyperactivity (Huang, Y. P. etal. (1988), J Immunol 141:827-833). Previously, we attempted to inhibitspontaneous Ig production in SLE with IL-2, but the results, howeverwere extremely variable. While we observed strong inhibition in somecases, in others IL-2 markedly increased Ig production. We believe thatthe timing and the cytokine millieu explains the more consistentinhibition observed in this study. Here the IL-2 and TGF-β were presentonly during the initial 72 hours of culture rather than the entireculture period. Enhancement of Ig synthesis in the latter case could beexplained by the positive effects of IL-2 on B cell differentiation(Coffman, R. L. et al. (1988), Immunol Rev 102:5-28). IL-2 candown-regulate antibody production by several mechanisms. In addition tothe TGF-β circuit described in the report, IL-2 induced inhibition canoccur by up-regulation of IFN-γ (Noble, A. et al. (1998), J Immunol160:566-571), or by cytolytic mechanisms (Stohl, W. et al. (1998), JImmunol 160:5231-5238; Esser, M. T. et al. (1997), J Immunol158:5612-5618). Previously, we had investigated the regulatory effectsof NK cells on antibody synthesis and reported that while the directeffect of NK cells is to up-regulate IgG synthesis (Kinter, A. et al.(1995), Proc Natl Acad Sci USA 92:10985-10989), these lymphocytes havethe opposite effect when cultured with CD8+ T cells in healthy subjects(Gray, J. D. et al. (1994), J Exp Med 180:1937-1942). In SLE patients,however, the combination of CD8+ T cells and NK cells enhanced IgGproduction (Linker-Israeli, M. et al. (1990), Arthritis Rheum33:1216-1225). This was again observed in the present report. While inthe normal subject the addition of NK cells to CD8+ T cells markedlyinhibited anti-CD2 stimulated IgG synthesis, the opposite was observedin SLE. From studies of normals we had learned that NK cell-derivedTGF-β induced co-stimulated CD8+ T cells to down-regulate IgG and IgMproduction (Gray, J. D. et al. (1998), J Immunol 160:2248-2254). In thisstudy IL-2 and TGF-β induced moderate suppressive activity by CD8+ Tcells. It is likely, therefore, that in SLE at least one way that IL-2and TGF-β inhibit B cell activity is by generating regulatory T cells.In addition, other lymphocyte populations treated with these or othercytokines may also down-regulate B cells activity in SLE.

Example 2 The Correlation of TGF-β Production to Disease Activity andSeverity

Having shown that the lymphocyte production of the total and activeforms of TGF-β is decreased, we next asked whether these defectscorrelate with disease activity and/or severity. TGF-β1 production byblood lymphocytes from 17 prospectively studied SLE patients wascompared with 10 rheumatoid arthritis (RA) patients and 23 matchedhealthy controls. In RA the levels of active TGF-β1 were lower thancontrols, but not deceased to the extent found in SLE. Levels ofconstitutive and anti-CD2 stimulated active TGF-β1 detected in picomolaramounts were markedly reduced in 6 untreated patients hospitalized withrecent onset, very active and severe SLE and similarly reduced in 11patients with treated, less active disease. thus, decreased productionof active TGF-β1 did not correlate with disease activity. By contrast,decreased production of total TGF-β1 inversely correlated with diseaseactivity. Thus it appears that although impaired lymphocyte secretion ofthe latent precursor of TGF-β1 may result as a consequence of diseaseactivity, the ability to convert the precursor molecule to its activeform may be an intrinsic cellular defect. Insufficient exposure of Tcells to picomolar concentrations amounts of TGF-β1 at the time they areactivated can result in impaired down-regulation of Ig synthesis. Thus,decreased lymphocyte production of active TGF-β1 in SLE can contributeto B cell hyperactivity characteristic of this disease.

Methods

Study Subjects

Seventeen subjects with a diagnosis of SLE who fulfilled the AmericanCollege of Rheumatology criteria for the classification of SLE (Tan, E.M. et al. (1982), Arthritis Rheum 25:1271-1277), 10 subjects with RA whofulfilled the ACR 1987 revised criteria for the classification of RA(Arnett, F. C. et al. (1988), Arthritis Rheum 31:315-324), and 23healthy donors were studied. The SLE group consisted of 15 women and 2men (15 Hispanic, 1 African American, 1 Asian). The mean age was 34.5years (range, 20-75 years). Six patients were hospitalized, and 11 wereattending an outpatient clinic. All of the hospitalized patients wereuntreated before admission and were studied before they received theirfirst dose of corticosteroids. Outpatients were receiving less than 20mg of prednisone, and none were receiving cytotoxic drugs. Diseaseactivity was assessed with the SLAM (Liang, M. H. et al. (1989),Arthritis Rheum 32:1107-1118) and SLEDAI (Bombardier, C. et al. 1992),Arthritis Rheum 35:630-640) indices with mean values of 6.6 and 7.6,respectively. The RA group consisted of 9 women and 1 man (9 Hispanic, 1Asian). The mean age was 50.9 years (range, 39-67 years). All of thepatients were attending the outpatient clinic and had mild to moderatelyactive disease. The mean duration of disease was 9.5 years. One patientreceived myochrysine, 3 patients received prednisone (1, 1 and 20mg), 3patients received methotrexate, and one patient received sulphasalazine.Healthy donors served as controls and were matched as closely aspossible for age, sex, and ethnic groups.

TABLE 5 Clinical Characteristics of Two Groups of SLE PatientsHospitalized Outpatient Clinical Data (n = 6) (n = 11) p Value Age 26.838.6 1.037 Sex (F/M) 6/0 9/2 Ethnic Group (H/AA/A) 5/0/1 10/1/10 DiseaseDuration (yr) 0.71 8.25 0.051 Disease Activity SLAM 13.3 2.9 0.014SLEDAI 15.7 4.1 0.006 Prednisone dose (mg/day) 41.2 7.8 0.008 ActiveRenal disease 83% 9% 0.028 Hemolytic Anemia 67% 9% 0.064 Anti-DNA(titer) 466.7 33.0 0.064 C3 47.5 98.6 0.008 C4 13.7 18.6 0.127

Reagents

Antibodies used were supernatants of hybridomas secreting anti-CD2(OKT11, American Type Culture Collection (ATCC), Rockville, Md., and GT2made available by Dr. Alain Bernard, Nice, France). A monoclonalantibody recognizing TGF-β isoforms 1, 2 & 3 (1D 11), an antibodyagainst TGF-β isoforms 2 & 3 (3C7), and rTGF-β2 were kindly provided byDr. Bruce Pratt (Genzyme Pharmaceuticals, Farmington, Mass.).

Isolation of Blood lymphocytes

Peripheral blood mononuclear cells (PBMC) were prepared from heparinizedvenous blood by Ficoll-Hypaque (Pharmacia, Piscataway, N.J.) densitygradient centrifugation using methods described previously (Ohtsuka, K.et al. (1998), J Immunol 160:2539-2545). The mononuclear cells werewashed in PBS with 5 mM EDTA (Life Technologies, Grand Island, N.Y.) toremove platelets, which are a rich source of TGF-β. Peripheral bloodlymphocytes (PBL) were separated from PBMC by centrifugation through acontinuous Percoll (Pharmacia) density gradient. The percentage ofmonocytes remaining in the high density, lymphocyte-enriched fractionwas somewhat higher in SLE (8.5% vs 4.3%).

Cell Culture Procedures

Procedures for cell cultures have been described previously ((Ohtsuka,K. et al. (1998), J Immunol 160:2539-2545). In brief, 1×10⁵ of thelymphocytes were added to the wells of 96-well flat bottom microtiterplate (Greiner Rocky Mountain Scientific, Salt Lake City Utah). Thecultures were carried out in AIM-V serum free medium (LifeTechnologies), since serum contains significant amount of latent TGF-β.Anti-CD2 was used at the optimal concentrations to induce TGF-βproduction (GT2 1:40 and T11 1:80) hybridoma culture supernatants.Previous studies have revealed that anti-CD2 strongly stimulates PBL toproduce TGF-β (Gray, J. D. et al. (1998), J Immunol 160:2248-2254).

TGF-β Assay

Mink lung epithelial cells (MLEC) which had been transfected with anexpression construct containing a plasminogen activator inhibitor(PAI-1) promoter fused to luciferase reporter gene were kindly providedby Dr D. B. Rifkin, New York, N.Y. MLEC at 2×10⁴/well were incubatedwith 200μl supernatants for 18 h at 37° C. To assay for luciferaseactivity, MLEC were lysed by a cell lysis reagent (AnalyticalLuminescence, Ann Arbor, Mich.). Cell lysates were then reacted withassay buffer and luciferin solution (both from Analytical Luminescence)immediately before being measured in a luminometer (Lumat, BertholdAnalytical Instruments Inc., Nashua, N.H.). To measure total TGF-βactivity, samples were heated at 80° C. for 3 minutes to release theactive cytokine from the latent complex. Active TGF-β activity wasmeasured without heating of supernatants. In all assays, severalconcentrations of rTGF-β were included to generate a standard curve. Thevariability of replicate cultures was less than 10 percent (Ohtsuka, K.et al. (1998), J Immunol 160:2539-2545).

Statistical Analysis

The significance of the results was analyzed using the Mann-Whitney testand Spearman rank correlation performed using GBSTAT software(Professional Statistics and Graphics Computer Program, DynamicMicrosystems Inc., Silver Spring, Md.).

We measured constitutive and stimulated TGF-β1 produced by PBL frompatients with SLE or RA, and compared these values with those fromnormal controls. The cytokine detected in culture supernatants wasneutralized by a mAb recognizing isoforms 1, 2, & 3, but not by oneagainst isoforms 2 & 3, a result confirming the production of TGF-β1.Compared to normal controls, constitutive production of active TGF-β1was significantly decreased in SLE (14±5 vs 56±21 pg/ml, p=0.02, FIG.5). Anti-CD2 stimulated active TGF-β1 was also decreased (87±22 vs399±103 pg/ml, p=0.003). In RA, the mean value for constitutive TGF-β1was similar to that of SLE (19±5 pg/ml) and after stimulation byanti-CD2 was intermediate between normal and SLE (197±54 pg/ml).

Constitutive total TGF-β1 produced by lymphocytes was also decreased inSLE in comparison with the normal group (286±82 vs 631±185 pg/ml,p=0.05). The value in RA was intermediate between normal and SLE(435±161 pg/ml). Following the addition of anti-CD2, total TGF-β1increased in SLE somewhat more than in normal controls so that thedifferences were not statistically significant. Values in the RA groupwere again intermediate between the normal and SLE group.

To look for a possible relationship between decreased levels of TGF-β1and disease activity, we compared hospitalized SLE patients with thoseseen in the outpatient clinic. The clinical characteristics of these twogroups are summarized in Table 5. Those that were hospitalized wereyounger; 5 of 6 had symptoms for less than 3 months; they had markedlyactive disease; and most had severe SLE with nephritis and/or hemolyticanemia. The outpatient group by contrast, had chronic disease which hadbecome less active following treatment. Notwithstanding this markeddifference in disease heterogeneity, duration, activity, and severity,both constitutive and stimulated active TGF-β1 production weresignificantly decreased in both groups in comparison with normalcontrols (Table 6).

TABLE 6 Comparison of TGF-β1 Production by Lymphocytes from Two Groupsof Patients with SLE* SLE Normal Group 1 Group 2 (n = 23) (n = 6) (n =11) Active TGF-β1 (pg/ml) Constitutive 56 ± 21  21 ± 14†  10 ± 4† CD2stimulated 399 ± 103 117 ± 52†  70 ± 19‡ Total TGF-β1 (pg/ml)Constitutive 631 ± 185 132 ± 44† 365 ± 120 CD2 stimulated 771 ± 136 226± 74† 667 ± 166 *PBL 1 × 10⁵/well were cultured for 48 h, and thesupernatants were tested for TGF-β1.

When we looked for correlations between levels of active and totalTGF-β1 with disease activity, there was a significant negativecorrelation between anti-CD2 stimulated production of total TGF-β1 andthe SLEDAI (r=−0.55, p=0.03, but not the SLAM index (−0.43, p=11). TheSLEDAI index is weighted for central nervous system involvement andrenal disease. Thus, an impaired capacity for lymphocytes to secrete theprecursor form of TGF-β1 appears to be associated with severe disease.The Levels of active TGF-β1 did not correlate with disease activity.

The principal finding in this example is that decreased production ofactive TGF-β1 in SLE does not correlate with disease activity orseverity. Decreased amounts of constitutive and stimulated active TGF-β1were found in both patients with recent onset and established disease.Moreover, the values did not correlate with activity, as measured by theSLAM and SLEDAI indices, or severity as assessed by vital organinvolvement. However, while total TGF-β1 production was also decreasedin SLE, this defect appeared to correlate with disease activity. It wasfound chiefly in hospitalized SLE patients. The finding that totalTGF-β1 production correlated most strongly with the SLEDAI index, whichis weighted for major organ system involvement, also suggests arelationship with disease severity.

This study also included a control group of RA patients whose diseaseactivity was comparable to SLE patients with established disease.Although TGF-β1 values in the RA group was somewhat less than the normalcontrols, with the exception of constitutive active TGF-β1, themagnitude of the defect was not as marked as in SLE and was notstatistically significant

Previously, we have documented that NK cells are the principallymphocyte source of TGF-β and the only lymphocyte population toconstitutively produce this cytokine in its active form (Gray, J. D. etal. (1998), J Immunol 160:2248-2254). It was of interest, therefore, tofind that constitutive production of NK cell-derived TGF-β was decreasedin SLE. We also learned that both IL-2 and TNF-α could enhance theproduction of active TGF-β. Production of both of these cytokines aredecreased in SLE (Gray, J. D. et al. (1994), J Exp Med 180:1937-1942).However, in most patients exogenous IL-2 and TNF-α could not restoreTGF-β production to normal (Example 2). IL-10 production is increased inSLE (Llorente, L. et al. (1993), Eur Cytokine Network 4:421) andcorrelations between elevated levels and disease activity have beenreported (Housslau, F. A. et al. (1995), Lupus 4:393-395; Haglwara, E.et al. (1996), Arthritis Rheum 39:379). IL-10 can inhibit IL-2, TNF-αand TGF-β production (Example 2 and Moore, K. W. et al. (1993), Ann RevImmunol 11:165-190). The findings that production of active TGF-β isdecreased in patients with mild as well as active disease, and that wecould only partially reverse the production defect by antagonizing IL-10(Example 2), suggests that increased IL-10 production, by itself, cannotaccount for decreased lymphocyte production of active TGF-β1 in SLE.Several mechanisms are probably involved. It is likely that one or moredefects in the extracellular conversion of the latent precursor to themature, active form may explain this abnormality.

Although TGF-β has well documented inhibitory properties on lymphocyteproliferation and effector cell function (Letterio, J. J. et al. (1998),Ann Rev Immunol 16:137-162), stimulatory properties have also beenreported (Lee, H. M. et al. (1991), J Immunol 151:668-677). TGF-βmodulates cytokine production by stimulated T cells as well asupregulating its production. In mice, TGF-β1 selectively activates CD8+T cells to proliferate (Lee, H. M. et al. (1991), J Immunol151:668-677), and augments the maturation of naive cells to memory Tcells (Lee, H. M. et al. (1991), J Immunol 147:1127-1133). in humansTGF-β1 is a potent inducer of effector T cells (Cerwenka, A. et al.(1994), J Immunol 153:4367-4377). While large (nanogram/ml) quantitiesare required for immuno-suppressive effects, we have shown that onlysmall (picogram/ml) quantities are needed to co-stimulate CD8⁺ T cellsfor down-regulatory effects on antibody production (Gray, J. D. et al.(1998), J Immunol 160:2248-2254).

These studies suggest, therefore, that while impaired lymphocytesecretion of the latent precursor of TGF-β1 may result as a consequenceof disease activity, decreased active TGF-β1 production in SLE is morecomplex and may result from several different mechanisms. We haveproposed that programming naive T cells to down-regulate antibodyproduction requires the presence of pg/ml quantities of active TGF-β atthe time they are activated and have evidence to support this suggestion(Gray, J. D. et al. (1998), J Immunol 160:2248-2254). Therefore, a lackof picomolar amounts of active TGF-β in the local environment at acritical time could possibly account for ineffective T cell regulatoryfunction to control B lymphocyte activity in SLE.

Example 3 Treating SLE with Mitogens

In this example, IgG production is down regulated by treating the cellswith an inhibitory composition comprising a mitogen such as Con A. Thecells are prepared as outlined in the above examples, and then they areincubated with mitogens to augment the population of cells that downregulate antibody production. In this case, the cells are incubated withphysiological concentrations of Con A for 4 to 72 hours using standardincubation techniques. The concentration of Con A used can range fromabout 0.01 to about 10 micrograms/ml with 1 microgram/ml being presentlypreferred. Con A is available from Sigma (St. Louis, Mo.).

Although it is not known how the mitogens work, it is believed to inducethe production of TGFβ by monocytes in the PBMCs preparation, and theTGFβ then acts on T cells to become antibody suppressor cells.

The cells are then washed, if necessary, and transplanted back into thepatient.

Example 4 Treating Cells with a Mixture of Cytokines and Mitogens

In this example, IgG production is down regulated by treating the cellswith an inhibitory composition comprising a mixture of cytokine andmitogen. The cells are prepared as outlined in the above examples, andthen they are incubated with the mixture to augment the population ofcells that down regulate antibody production, such as physiologicalconcentrations of Con A, IL-2 and TGFβ, or Con A and IL-2, for 4 to 72hours using standard incubation techniques.

After the cells have been incubated with the cytokines and mitogen, thecells are then washed with HBBS to remove any cytokine and mitogen thatare in the solution. The cells are then suspended in 200-500 ml of HBBSand are reintroduced into the mammal.

What is claimed is:
 1. A method for treating an autoimmune disorder in apatient comprising: a) removing peripheral blood mononuclear cells(PBMC) from said patient; b) treating said cells with an inhibitorycomposition for a time sufficient to suppress immunoglobulin production;and c) reintroducing said cells to said patient.
 2. A method accordingto claim 1 wherein said inhibitory composition comprises IL-2.
 3. Amethod according to claim 1 wherein said inhibitory compositioncomprises a mixture of IL-2 and TGF-β.
 4. A method according to claim 1wherein said inhibitory composition comprises a CD2 activator.
 5. Amethod according to claim 1 wherein said autoimmune disorder is systemiclupus erythematosus (SLE).